Marine Metagenomics
Marine Metagenomics The term metagenomics was proposed by Handelsman to describe the entire set of sequences of organisms living in a defined habitat. [5] With specific regard to marine metagenomics, the techniques and methods are mostly developed from the study of soil metagenomics.[3] The particular study emerged as a combination of extractoin and digestion of bacterial DNA from the soil and, likewise, the general methods of marine metagenomics function. [4] Origin & Importance of Marine Metagenomics The initial goal of marine metagenomics was to catalogue the vast microbial diversity of the oceans, with the goal of providing acces to the functions of previoiusly hidden essential microbes and their role in the main geochemical cycles. The first large-scale attempts at this was the metagenome investigation lead by C. Venter and team in the Sargasso Sea [1] and the Sourcer II expedition. [2] The principle advantage is that it allows the currant limits of culture-dependent methods to be bypassed. Specifically, marine environments are particularly diverse and the exploration thereof offers untapped resources for biotechnology. Origin of Pyrosequencing Most recently, the majority of large-scale metagenomic studies rely on pyrosequencing apporaches. Traditional Sanger sequencing relies on the culturing and cloning of an organism, which is unsuitable for metagenomic studies.The Roche 454 platform is most often used to deliver high throughput with the largest reads of currently available next-generation sequencing platforms (~450 bp). Becasue shorter fragment lengths may lack en ough sequence-specific information for organism identification, the longer fragments are essential. The longer fragment also aids in distinguishing and classifying unknown sequences from many different organisms in a mixed sample. [8] The method of pyrosequencing is also considerably cheaper and faster than the Sanger method becuase it lacks the requirement for cloned DNA libraries, which also avoids the conflict of cloning biases. [7] Outline of Pyrosequencing The widely applicable method of pyrosequencing is particularly useful for the detailed characterization of nucleic acids. Of the many potential advantages, accuracy, flexibility, parallel processing, and potential to automate pyrosequencing is a useful addition to the tool kit of metagenomic analysis. [7] (1) The detection of released pyrophosphate (PPi) during DNA synthesis is essential to pyrosequencing. In a cascade of enzymatic reacions a proportional quantity of visible light is generated from the number of nucleotides within the sample. (2) The cascade begins with a nucleic acid polymerization in which inorganic PPI is released due to nucleotide incorporation by polymerase. The released PPI is then converted by sulfurylase to ATP by ATP. This energy is necessary to luciferase to oxidize luciferin, which generates light (exothermic reaction). (3) The nucleic acid molecule can be either RNA or DNA and because the added nucleotide is known, the sequence of the template can be determined. [7] Further Application and Study http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005299 References 1 Venter J.C. et. al. Environmental genome shotgun sequencing of the Sargasso Sea. Science. 2004. 304(5667): 66-74 2 Rusch D.B. et. al. The Sourcer II global ocean sampling expedition: northwest Atlantic through eastern tropical Pacific. PLoS Biology. 2007. 5(3). 3 Schneegurt M.A. et. al. Direct extraction of DNA from soils for studies in microbial ecology. Curr. Issues Mol. Biol. 2003. 5: 1-8 4 Torsvik V.C. Isolation of bacterial DNA from soil. Soil Biol. Biochem. 1980. 12: 15-21 5 Handelsman J. et. al. Molecular biological access to the chemistry of unknown soil microbes: a new fronteir for natural products. Chem. Biol. 1998. 5(10) 6 http://en.wikipedia.org/wiki/Pyrosequencing 7 Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res. 2001. 11(1): 3-11 8 Citig D. Pyrosequencing sheds light on DNA sequencing. ''Genome Res. ''2001. 48(5): 361-365 9 Woyke T. et. al. Assembling the marine metagenome, one cell at a time. PLoS ONE. 2009. 4(4)